Stable provitamin d composition



Patented Jan-6,1948 2 434 015 V UN TED STATES PATENT OFFlCE STABLE PROVITAMIN n COMPOSITION Hans R. Rosenberg and Warren W. Woessner, Wilmington, Del., assignors to E. I. du Pont de Nemours & Company, Wilmington, Del., a corporation of Delaware No Drawing. Application November 21, 1942, Serial No. 466,496

1 1 Claim. (Cl. 167-81) This invention relates to stable provitamin D crystals containing approximately 50% 7-dehycompositions and more particularly refers to drocholesterol and approximately 50% cholescompositions containing in intimate contact terol. Additional embodiments of this invenprovitamin D and a stabilizing agent or agents, tion are concerned with the employment of the aswell as processes for the production and use foregoing compositions in antirachitic activation of such compositions. processes wherein they are subjected to the action It is known that many provitamins D d teriof ultra-violet light or some other activating orate rapidly upon exposure to air or other natmural influences. Some provltamins D are much The invention y be more readily understood more susceptible to these deteriorating influences m by a Consideration Of the win illustrative than others. For example, 7-dehydrocholesterol exampleswill deteriorate much more rapidly upon exposure Example I to air than will ergosterol. Nevertheless all Mixtures of pure 7-dehydrocholesterol and gggggg g 2 :;s s'lfgz zfi i g n cholesterol in four different proportions were dis- Many agtelgpts have been a i is; g g? solved in ethanol and crystallized from this solvent. The recovered preparations, beautifully Z225 gi i i g of i have white and crystalline, were dried and a spectrotime-c onsui nii ig a fi l i ):gensive r ifi taii ll l m illi scopic gg f g $5 3 n tively, 1%, an o e y r instances to produce the desired degree of stabicholesterol. Conversely, they contained, respec ft i s an object of this invention to overcome tively' 1 t i choleistergl. the foregoing disadvantages and many others gf f i jgf fi xg i sf gfig gf 53 i which-airway or indirectly result m P A tions of storage pure 7-dehydrocholesterol will 323 2; ig :3 DrOduCebpmXitaIEIEISQm Such 25 show evidence of deterioration in less than two riods of t lme and fnfi lz wid elg ilarifi ng c n d i: Flays' The presfem sampliiremmedficfianfied tions without serious deterioration or contamina g ig igg ggz Zi ia? g 2 x 3 gg fi tion. A still further object is to produce stable teml began to Show Slight d e of yellowing provitamin D compositions which facilitate the At the end f three Weeks the sample with e recover? of unactivated. Q D after P next lowest amount of cholesterol had begun to compositions have been sublected to antirachitic Show some Slight discoloration However, the

activation Additional objects will become two samples with the higher cholesterol content parent from a consideration of the following remained complete]y unchanged even ft fi spec fication and claim. 5 weeks, Storage These objects are attained in accordance with when the above example was repeated, the present invention wherein a composition stituting for cholesterol the inactivatable sterols produced containing at least 30% provitamin D from mussels. substantially similar results were in intimate contact with a sumcient amount of obtained anti-rachitically inactivatable sterols to stabilize 40 said provitamin D. In a more restricted sense Example H this invention is concerned with a stable pro- Freshly gathered mussels of the species vitamin D particularly adapted for antirachitic Modiolus demissus (Dillwyn) were subjected to activation comprising amass of crystals containsteam for a period of 5 to 10 minutes. The

ing at least 30% provitamin Band at least 20% 46 meat was then separated from the shells and was of antirachitically inactivatable sterols. In a saponified with alcoholic potassium hydroxide in still more restricted sense this invention is conan amount of about one part of alcohol and onecerned with crystals of the foregoing type wheretenth part of potassium hydroxide for one part in the stabilizing sterol is cholesterol. In a still of the mussel meat. The saponification was more restricted sense this invention pertains to 0 carried out at the reflux temperature and was stable provitamins D of the foregoing type continued over a period of from two to'ten hours. wherein the provitamin D is 7-dehydrocholesterol After completion of the saponification procedure and the stabilizing sterol is cholesterol. In its two parts of water were added per part of the preferred embodiment this invention is directed saponlfication mass. The mixture was then exto a stable provitamin D comprising a mass of tracted with ether and the ether solution washed neutral with water and dried over a desiccating agent. The ether was then distilled oil and the residue was dissolved in -20 times its weight of alcohol and the sterol-provitamin D mixture was allowed to crystallize. After recrystallization from alcohol or another suitable solvent, the sterol mixture contained 43% of provitamin D, and 57% of inactivatable sterols.

This sterol mixture was essentially stable on storage under ordinary conditions. On the other hand the provitamin D isolated from mussels of the species Modiolus demissus (Dillwyn) proves to be unstable when present in a concentration of 85% or more in mixture with or less of in- Mussels of the genus, Modiolus, were subjected to saponification in alcoholic alkali. The sterols isolated as described in Example II contained according to spectroscopic examination 45-50% of provitamin D, and'55 to 50% respectively of inactivatable sterols.

2.8 parts of this sterol mixture were dissolved in 1250 parts of ether which was freshly distilled over sodium. This solution was subjected to rradiation with ultraviolet light according to the well known procedure. After completion of the irradiation, the ether was distilled off and from the residue the unchanged provitamin D together with the inactivatable sterols was crystallized by means of alcohol. The crystals were filtered off from the mother liquors which contained the vitamin D. Corn oil was added to these mother liquors and the alcohol distilled off under vacuum leaving the vitamin D dissolved in oil.

The crystals containing provitamin D were found to be extremely stable on storage for prolonged periods.

Emample IV The inactivatable sterols isolated from a vegetable oil were recrystallized once from ethyl alcohol. These phytosterols, together with an equal amount of 7-dehydrocholesterol, were dissolved in hot alcohol and allowed to crystallize in the cold. The recovered mixture of sterols containing approximately 50% of 7-dehydrocholesterol was found to be stable on storage and to give excellent yields of vitamin D on antirachitic activation.

The great utility of this invention is apparent when it is applied to the activation of unstable provitamins. For example, when 7-dehydrocholesterol alone in a solution of ethyl ether is irradiated with ultra violet light great care is required to realize the full yield of vitamin D; especially is this the case in large scale or commercial operations. In the latter case it is difiicult to recover all of the unchanged 7-dehydrocholesterol and, in the manipulations necessary to this recovery step and in the time which it is necessary to hold the recovered fractions before they can be subjected to further irradiation, deterioration and losses take place. If, however, the 7-dehydrocholesterol is irradiated in the presence of an approximately equal quantity of cholesterol or other inactivatable sterol the operations are carried out with greater ease and increased over-all yields of vitamin D are obtained. Since the cholesterol has only a slight general absorption in the region of the activating wavelengths of light it does not interfere with the irradiation of the provitamin yet its presence greatly facilitates the recovery of unchanged 7- 4 dehydrocholesterol by aiding it to precipitate in crystalline form from the solvents used in the recovery steps. Further, of course, all recovered fractions are crystalline mixtures of 7-dehydrocholesterol and cholesterol and are stable and may be held without deterioration or change till they can be reirradiated. By such a procedure, at little or no cost, increased amounts of vitamin D are secured from the provitamin.

It is to be understood that the foregoing examples are illustrative merely of a few of the many embodiments of the present invention. They may be varied widely with respect to the individual components, the proportions thereof, and their method of production and use without departing from the scope of this invention.

This invention is applicable to the stabilization of all provitamins D. While 7-dehydrocholesterol and mussel provitamin D have been referred to in the examples, it is to be understood that in place thereof or in addition thereto other provitamins D may be employed. A representative few of these provitamins D are ergosterol, epi-ergosterol, Z-dehydrosltosterol, 7-dehydrostigmasterol, 'l-clehydrocampesterol, epi-7-dehydrocholesterol, ZZ-dihydroergosterol, etc. These provitamins may be derived from natural sources or they may be synthesized.

In place of, or in addition to, the inactivatable sterols previously described other antirachitically inactivatable sterols may be employed as stabilizing agents. As mentioned heretofore, these inactivatable sterols may be used alone or two or more of them may be used in admixture with one another. Likewise, they may be derived from natural sources or they may be synthesized. An illustrative few of the inactivatable sterols satisfactory for use herein are cholesterol, inactivatable sterols from mussels and other invertebrata, sitosterol, stigmasterol, brassicasterol, ostreastero1, campesterol, fungisterol, zymosterol, clionasterol, fucosterol, dihydrocholesterol, coprosterol, sitostanol, and the like.

It is to be understood that since cholesterol and other inactivatable sterols appear to have the most pronounced stabilizing effect on '7-dehydrocholesterol and on mussel provitamin D these are the preferred embodiments of this invention, partlcularly the mixture of cholesterol and 7-dehydrocholesterol.

The amount of stabilizing agent used will vary widely depending upon the particular provitamin D to be stabilized and the manner in which it is to be stored or employed. Amounts of stabilizing agents ranging from about 25% to more than 100% of the amount of provitamin D have been found to give excellent results. As a general rule it is advisable to use for optimum results approximately equal amounts of the stabilizing agent and the provitamin D to be stabilized, This is particularly true when l-dehydrochloresterol is the provitamin D and cholesterol is the stabilizing agent.

Crystalline mixtures of the provitamin D and the stabilizing agent may readily be prepared by dissolving them in a common solvent and permitting them to crystallize from this solvent in accordance with standard crystallization technique. The resulting crystals may be dried and stored for extended periods of time without noticeable deterioration. i

When these crystalline stabilized mixtures are subjected to antirachitic activation they are found to dissolve readily in the organic solvents commonly used for this purpose. During activation the presence of the stabilizing agent appears to exert a beneficial efiect upon the provitamin D undergoing activation. When the activation is completed the stabilizing agent permits the unactivated provitamin D to be separated'readily from the vitamin D present in the solution. This may be explained on the theory that the stabilizing agent and the provitamin D crystallize readily in the same crystal structure thereby accomplishing the dual purpose of facilitating separation of vitamin D from provitamin D and stabilizing the latter. In the absence of this stabilizing agent it wouldbe extremely difiicult to separate the unactivated provitamin D from the vitamin D present in the solution. In fact, attempts to separate completely these two components generally result in appreciable destruction or loss of the provitamin D.

By means of the present invention stable provitamin D compositions may be obtained. These compositions can be stored for long periods of time under widely varying conditions without appreciable deterioration or contamination. Likewise, they are particularly adapted for use in antirachtic activation processes since they not only stabilize the provitamin D during its subjection to activating influences-but they also facilitate separation of the unactivated provitamin D from the vitamin D at the termination of the activating process. It is well known that in the commercial antirachitic activation of any provitamin D it is highly uneconomical to secure complete transformation of the provitamin on a single exposure to the activating agent. Hence, in carrying out such an operation it is always necessary to recover unchanged provitamin by separating it from the vitamin D and other activation products. Consequently, any expedient which facilitates such operation and increases the efllciency thereof is of considerable importance. Furthermore, these stabilizing agents and in particular cholesterol do not absorb any appreciable amount of activating energy, so

VREFERENCES, CITED The following references are of record in the fileof this patent:

UNITED STATES PATENTS Number Name Date 2,163,659 Boer et al June 27, 1939 1,871,136 Steenbock Aug. 9, 1932 1,902,745 Windaus Mar. 21, 1933 2,234,554 Elley Mar. 11, 1941 2,216,719 Boer et a1. Oct. 8, 1940 2,257,176 Linsert Sept. 30, 1941 2,264,320 Linsert Dec. 2, 1941 OTHER REFERENCES Koch, Chemical Abstracts, vol. 31, page 1075 (1937).

Koch et al., Journal of Biological Chemistry.

vol. 116 (Dec. 1936), pages 757 to- 768. 

